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J. Phycol. 42, 482–492 (2006) r 2006 Phycological Society of America DOI: 10.1111/j.1529-8817.2006.00210.x

DEFINING THE MAJOR LINEAGES OF RED (RHODOPHYTA)1

Hwan Su Yoon Department of Biological Sciences and Roy J. Carver Center for Comparative , University of Iowa, 468 Building, Iowa City, Iowa 52242, USA Kirsten M. Mu¨ller Department of Biology, University of Waterloo, Waterloo, ON, N2L 3G1 Robert G. Sheath Office of the Provost, California State University San Marcos, San Marcos, California 92096, USA Franklyn D. Ott 905 NE Hilltop Drive, Topeka, Kansas 66617, USA and Debashish Bhattacharya2 Department of Biological Sciences and Roy J. Carver Center for Comparative Genomics, University of Iowa, 446 Biology Building, Iowa City, Iowa 52242, USA

Previous phylogenetic studies of the Rhodophyta dospora,andRufusia). We also describe a new have provided a framework for understanding red , Rhodellales, and a new , Rhodellaceae algal phylogeny, but there still exists the need for a (with Rhodella, Dixoniella, and Glaucosphaera). comprehensive analysis using a broad sampling of Key index words: ; Compsopogono- taxa and sufficient phylogenetic information to phyceae; ; Florideophyceae; clearly define the major lineages. In this study, we Porphyridiophycae; red algal lineages; Rhod- determined 48 sequences of the PSI P700 chl a ellophyceae; Rhodophyta; apoprotein A1 (psaA) and rbcL coding regions and established a robust red algal phylogeny to identify Abbreviations: BPP, Bayesian posterior probabili- the major . The tree included most of the lin- ties; ML, maximum likelihood; MP, maximum par- eages of the Bangiophyceae (25 genera, 48 taxa). simony; PsaA, PSI P700 apoprotein Seven well-supported lineages were identified with A1; PsaB, PSI P700 chlorophyll a apoprotein A2; this analysis with the Cyanidiales having the earliest PsbA, PSII reaction center protein D1; PsbC, PSII divergence and being distinct from the remaining 44 KD apoprotein; PsbD, PSII D2 reaction center taxa; i.e. the Porphyridiales 1–3, , Flori- protein; TBR, tree bisection-reconnection deophyceae, and . We also analy- zed data sets with fewer taxa but using seven pro- teins or the DNA sequence from nine genes to resolve inter- relationships. Based on all of The (Rhodophyta) are a distinct eukaryotic these analyses, we propose that the Rhodophyta lineage whose members are united in phylogenetic anal- contains two new subphyla, the yses of nuclear, , and mitochondrial genes (Fresh- with a single , the Cyanidiophyceae, and the water et al. 1994, Ragan et al. 1994, Van de Peer and De Rhodophytina with six classes, the Bangiophyceae, Wachter 1997, Burger et al. 1999, Yoon et al. 2002b, , Florideophyceae, Por- 2004). Rhodophytes lack chl b and c but contain all- phyridiophyceae classis nov. (which contains Por- ophycocyanin, , and in the phyridium, Flintiella,andErythrolobus), Rhod- of on unstacked . The ellophyceae, and Stylonematophyceae classis nov. plastid in these taxa is bound by two membranes and (which contains , Bangiopsis, Chroodacty- produces floridean starch that is deposited in the cyto- lon, Chroothece, Purpureofilum, Rhodosorus, Rho- plasm. All members of this group lack flagella and cent- rioles in all stages of the history (Gabrielson et al. 1990, Graham and Wilcox 2000). It is believed that the 1Received 13 September 2005. Accepted 27 December 2005. red algal plastid originated from a cyanobacterial pri- 2Author for correspondence: e-mail debashi-bhattacharya@ mary endosymbiosis and this organelle shares a com- uiowa.edu. monancestrywithgreenandglaucophytealgae

482 THE MAJOR LINEAGES OF RED ALGAE 483

(Bhattacharya and Medlin 1995, Delwiche et al. 1995, MATERIALS AND METHODS Cavalier-Smith 1998, McFadden 1999, Bhattacharya et al. 2004, Rodrı´guez-Ezpeleta et al. 2005). These three sampling and sequencing. Forty-eight red algal taxa ‘‘primary’’ plastid-containing groups are considered to were used to infer the phylogeny of the Bangiophyceae (Ta- be taxonomically united in the Plantae (Cava- ble 1). The data set included all bangiophycean orders and 25 genera from the different phylogenetic lineages (Garbary lier-Smith 1998) or (Adl et al. 2005). and Gabrielson 1990, Mu¨ller et al. 2001). Our alignment also Traditionally, the Rhodophyta has been di- included 10 , two , and three cyano- vided into two classes (or subclasses), Bangiophyceae as the outgroup (Bhattacharya and Medlin 1995, (Bangiophycidae) and Florideophyceae (Florideo- Moreira et al. 2000). We obtained algal cultures from the phycidae) (Garbary and Gabrielson 1990). However, Culture Collection of Algae & (CCAP), Provasoli- recent studies have concluded that the Florideo- Guillard National Center for Culture of Marine Phytoplank- phyceae form a monophyletic group with the order ton (CCMP), the Dipartimento di Biologia Vegetale (DBV) culture collection at the University of Naples, the Sammlung Bangiales (Oliveira and Bhattacharya 2000, Mu¨ller von Algenkulturen (SAG) at the University of Go¨ttingen, and et al. 2001, Yoon et al. 2002b, Saunders and Hommer- the Culture Collection of Algae at the University of Texas at sand 2004). The Bangiophyceae, which has in the past Austin (UTEX). Some of the bangiophytes were collected in been divided into six orders (Bangiales, Cyanidiales, the field and/or maintained in the private collection of F. D. Ott. Compsopogonales, , Porphyridiales, The algal cells were frozen in liquid nitrogen and ground t and Rhodochaetales), is now considered to form a se- with glass beads using a glass rod and/or Mini-BeadBeater (Biospec Products Inc., Bartlesville, OK, USA). Total genomic ries of radiations that define the ancestral lineages of DNA was extracted using the DNeasy Mini Kit (Qiagen, the red algae (Gabrielson et al. 1985, Freshwater et al. Santa Clarita, CA, USA). PCR were carried out using specific 1994, Ragan et al. 1994). Comprehensive phylogenetic primers for each plastid gene (Yoon et al. 2002a). Because in- studies of the Bangiophyceae (Oliveira and Bhatta- trons were found in the psaA gene of some red algae, the RT- charya 2000, Mu¨ller et al. 2001, 2003, West et al. 2005) PCR method was used to isolate cDNA for these coding regions using the plastid and nucleus-encoded small subunit (H. S. Yoon et al. unpublished data). The PCR products were purified using the QIAquick PCR Purification Kit (Qiagen), (SSU) rDNA and plastid rbcL show furthermore that and were used for direct sequencing using the BigDyet Ter- the Porphyridiales are paraphyletic and comprise at minator Cycle Sequencing Kit (PE-Applied Biosystems, Nor- least three independent lineages. This result is gener- walk, CT, USA), and an ABI-3700 at the Roy J. Carver Center ally consistent with previous morphological studies for Comparative Genomics at the University of Iowa. Some that show great differences in plastid ultrastructure PCR products were cloned into the pGEM-T vector (Promega, and variable vegetative and reproductive anatomy Madison, WI, USA) before sequencing. (Gabrielson et al. 1985, 1990, Garbary and Gabrielson Phylogenetic analyses. We primarily used amino acid se- quences in the phylogenetic analysis in order to minimize 1990, Mu¨ller et al. 2001). However, all of these previ- potentially misleading phylogenetic signal because of DNA ous studies are characterized either by broad taxon mutation bias at the third positions of codons (Sanderson sampling with a single gene (Mu¨ller et al. 2001) or et al. 2000, Pinto et al. 2003) or because of heterogeneous narrow sampling with multiple genes (Yoon et al. codon usage (Inagaki et al. 2004). The protein sequences 2002b, 2004). For this reason, whereas the identity of were manually aligned using SeqPup (Gilbert 1995). Two the major red algal lineages has been relatively firmly data sets were used in the phylogenetic analyses and the alignments are available from D. Bhattacharya. In the first established, their interrelationships remain unclear. data set, we generated a concatenated alignment of seven Recently, Saunders and Hommersand (2004) pro- plastid-encoded proteins (7PEP; a total of 2564 aa): PsaA posed a new red algal taxonomic scheme based on (465 aa), PSI P700 chlorophyll a apoprotein A2 (PsaB, 422 previous molecular phylogenies and ultrastructural aa), PSII reaction center protein D1 (PsbA, 319 aa), PSII 44 characters (e.g. Golgi–ER association). Their taxo- KD apoprotein (PsbC, 334 aa), PSII D2 reaction center pro- nomic system is a large step forward but still contains tein (PsbD, 296 aa), RbcL (405 aa), and TufA (323 aa), from 16 bangiophytes, and from 15 outgroup taxa including green the paraphyletic class that includes and algae and . Because the rbcL both unicellular and pseudofilamentous forms (i.e. gene of the green and glaucophyte algae are of a cyanobac- Porphyridiales Kylin ex Skuja 1939, Stylonematales terial origin, whereas those in the red algae and red algal- K. Drew 1956, and ‘‘Porphyridiales 1’’ Mu¨ller derived are of proteobacterial origin (Valentin and et al. 2001). Zetsche 1990, Delwiche and Palmer 1996), the evolutionarily In this study, we determined 48 sequences from the distantly related green and glaucophyte rbcL sequences were PSI P700 chl a apoprotein A1 (psaA) and rbcL coding coded as missing data in the phylogenetic analyses. In the second amino acid data set, we combined only PsaA and RbcL regions with broad taxonomic sampling. We included (2PEP), and added 33 more bangiophytes to the alignment most of the lineages of Bangiophyceae (25 genera, 48 (now a total of 49 bangiophytes). The Cyanidiales, which is taxa) in the analyses because these taxa represent the the earliest diverging clade of red algae (Yoon et al. 2002b, ancestral pool of red algae. In addition, we analyzed 2004), was used as the outgroup for this data set. In addition data sets with fewer taxa but using seven proteins or to the amino acid data, we generated a DNA alignment that DNA sequence from nine genes to resolve inter-clade included nuclear SSU rDNA (18S; 1486 bp) and plastid SSU rDNA (16S; 1285 bp) sequences resulting in a nine-gene relationships. The combination of all of these phylo- DNA sequence data set (9GDS; 10,463 bp) that excluded genetic studies was then used to advance our under- the green and glaucophyte algae. Three additional Cyanidi- standing of the higher-level taxonomic relationships ales were included in the DNA alignment and used as within the red algae. the outgroup for this data set. 484

TABLE 1. Sample information and GenBank accession numbers for taxa included in the phylogenetic analyses.

Taxa Species Source rbcL psaA psaB psbA psbC psbD tufA 18S rDNA 16S rDNA

Rhodophyta, Bangiophyceae Bangiales atropurpurea SAG 33.94 FD Ott O458 AY119770 AY119698 AY391374 AY119734 AY876202 AY876227 AF545587 D88387 AF545616 (Roth) C Agardh (fresh-water) Bangia fuscopurpurea SAG 59.81 (marine)a AY119771 AY119699 (Dillwyn) Lyngbyea Bangia sp. (maxima Bolinas, CA DQ308423 DQ308441 form) leucosticta SAG 55.88 DQ308424 DQ308442 Thuret GenBank NC_000925 NC_000925 NC_000925 NC_000925 NC_000925 NC_000925 NC_000925 AF362362 NC_000925 (Roth) C. Agardh Compsopogonales Boldia erythrosiphon Black River, Ontario, Canada AF078121 Missing Herndon coeruleus SAG 36.94 FD Ott O798 AF087116 AY119701 AY391375 AY119737 AY876203 AY876228 AF545589 AF342748 AF1707139 (Balbis ex C. Agardh) Montagne C. coeruleus FD Ott O646 DQ308425 DQ308443 (syn. 5C. hookeri ) C. coeruleus FD Ott O1123 DQ308426 DQ308444

(syn. 5C. oishii) AL. ET YOON SU HWAN C. coeruleus Alabama, USA AF087115 DQ308445 (syn. 5Compsopogonopsis leptoclados) Erythropeltidales carnea UTEX LB 1425 FD Ott MO14 AF087118 AY119703 (Dillwyn) J. Agardh Erythrocladia irregularis UTEXLB1419FDOtt AF087117 DQ308446 Rosenvinge MO360 Rhodochaetiales Rhodochaete parvula UTEX LB 2715 AY119777 AY119707 AY391389 AY119743 Missing Missing AF545601 AF139462 AF545623 Thuret Cyanidiales merolae DBV 201 JAVA AY119765 AY119693 AY391376 AY119729 NC_004799 NC_004799 AF545590 AF441376 AF545617 Luca, Taddei et Varano Cyanidium caldarium RK1 (Shimotsuke, Japan) NC_001840 NC_001840 NC_001840 NC_001840 NC_001840 NC_001840 NC_001840 AB090833 NC_001840 (Tilden) Geitler Cyanidium caldarium DBV 019 SIPE AY541297 AY541281 (Tilden) Geitler Cyanidium sp. Monte Rotaro, Italy AY391368 AY391362 AY391377 AY391365 Missing Missing AY391371 Missing AY391359 Cyanidium sp. Sybil Cave, Naples, Italy AY391369 AY391363 AY391378 AY391366 AY876204 AY876229 AY391372 Missing AY391360 daedala IPPAS P508 AY541302 AY541283 Sentsova Galdieria maxima IPPAS P507 AY391370 AY391364 AY391379 AY391367 AY876205 AY876230 AY391373 AB090832 AY391361 Sentsova Galdieria partita IPPAS P500 AB18008 AY541284 Sentsova SAG 108.79 AY119767 AY119695 AY391380 AY119731 AY876206 AY876231 AF545591 AF342747 AF170718 (Galdieri) Merola Galdieria sulphuraria UTEX 2393 AF233069 AY541285 (Galdieri) Merola Galdieria sulphuraria- DBV 009 VTNE AY119768 AY119696 AY391381 AY119732 AY876207 AY876232 AF545592 Missing AF545618 DBV Galdieria sulphuraria- DBV 012 BNTE AY541310 AY541288 DBV Porphyridiales P1 Dixoniella grisea SAG 39.94, FD Ott O113 AY119773 AY119702 AY391383 AY119738 Missing Missing AF545595 L26187 AF545621 (Geitler) Scott, Broadwater, Saunders, Thomas et Gabrielson P1 Glaucosphaera vacuolata UTEX LB 1662 DQ308427 DQ308447 Korshikov TABLE 1 (Continued).

Taxa Species Source rbcL psaA psaB psbA psbC psbD tufA 18S rDNA 16S rDNA

P1 Rhodella maculata Evans CCMP 736 DQ308428 DQ308448 P1 Rhodella violacea SAG 115.79 AY119776 AY119706 AY391386 AY119742 DQ308461 DQ308462 AF545598 AF168624 AF545622 (Kornmann) Wehrmeyer P2 Bangiopsis subsimplex PR21 (Puerto Rico) AY119772 AY119700 AY391382 AY119736 Missing Missing AF545594 AF168627 AF545620 (Montagne) Schmitz P2 Chroodactylon ornatum SAG 103.79, FD Ott MO447 DQ308429 DQ308449 (C. Agardh) Basson (syn. 5C. ramosum) P2 Chroothece mobilis FD Ott O1363 DQ308430 DQ308450 Pascher et Petrova´ P2 Kyliniella latvica Skuja FD Ott O506 DQ308431 DQ308451 P2 Purpureofilum CCAP 1383/1 DQ308432 DQ308452 apyrenoidigerum West et Zuccarello P2 Rhodospora sordida UTEX LB 2616 FD Ott O1515 DQ308433 DQ308453

Geitler ALGAE RED OF LINEAGES MAJOR THE P2 Rhodosorus marinus SAG 116.79 FD Ott MO360 AY119778 AY119708 Missing AY119744 AY876208 AY876233 AF545595 AF342750 AF170719 Geitler P2 Rhodosorus sp. CCMP 1530 DQ308434 DQ308454 P2 Rufusia pilicola Wujek FD Ott O7031 DQ308435 DQ308455 et Timpano P2 SAG 2.94 AY119779 AY119709 AY391388 AY119745 Missing Missing AF545600 AF168633 AF170714 (Zanardini) Drew P2 S. alsidii UTEX LB 1957 DQ308436 DQ308456 (syn. 5Goniotrichum elegans) P3 Erythrolobus coxiae Baca, FD Ott O530 DQ308437 DQ308457 Wolf et Cox P3 E. coxae (syn. 5E. fottii) UTEX LB 2545 DQ308438 DQ308458 P3 Flintiella sanguinaria SAG 40.94 FD Ott O340 AY119774 AY119704 AY391384 AY119740 DQ308463 DQ308464 AF545596 AF342749 AF170719 Ott P3 Porphyridium SAG 1380-2 AY119775 AY119705 AY391385 AY119741 AY876209 AY876234 AF545597 AF168623 X17597 aerugineum Geitler P3 Porphyridium purpureum CCMP 1328 DQ308439 DQ308459 (Bory de Saint- Vincent) Ross in Drew et Ross (syn.5 P. cruentum) P3 Porphyridium sordidum FD Ott O250 DQ308440 DQ308460 Geitler Rhodophyta, Florideophyceae crispus Nova Scotia, Canada U02984 AY119710 AY391390 AY119746 AY876210 AY876235 AF545602 Z14140 Z29521 Stackhouse palmata (L.) Maine, USA U28421 AY119711 AY391391 U28165 AY876211 AY876236 AF545603 Z14142 Z18289 Kuntze Thorea violacea Bory de SAG 51.94 FD Ott O282 AF029160 AY119712 Saint-Vincent Arabidopsis thaliana (L.) GenBank NA NC_000932 NC_000932 NC_000932 NC_000934 NC_000935 X52256 Heynhold formosae GenBank NA NC_004543 NC_004543 NC_004543 NC_004543 NC_004543 NA Chaetosphaeridium GenBank NA NC_004115 NC_004115 NC_004115 NC_004115 NC_004115 NA globosum (Nordstedt) Klebahn Chlamydomonas GenBank NA NC_005353 NC_005353 NC_005353 NC_005353 NC_005353 NC_005353 reinhardtii Dangeard Chlorella vulgaris GenBank NA NC_001865 NC_001865 NC_001865 NC_001865 NC_001865 NC_001865 Beijerinck 485 GenBank NA NC_001319 NC_001319 NC_001319 NC_001319 NC_001319 NA 486 HWAN SU YOON ET AL.

All protein phylogenies were reconstructed under maxi- mum likelihood (ML) using proml in the PHYLIP V3.6b pro- gram package (Felsenstein 2002). The trees were inferred with the JTT þ G evolutionary model and global rearrangements with four random addition replicates (Jones et al. 1992). The a values for the g distribution for the different data sets were calculated using TREE-PUZZLE V5.2 (Schmidt et al. 2002). To assess the stability of monophyletic groups in the ML trees, we calculated bootstrap support values using PHYML V2.4.3 (Guindon and Gascuel 2003) and unweighted maximum par- simony (MP) using PAUP*V4.0b10 (Swofford 2004). We also A 18S rDNA 16S rDNA calculated Bayesian posterior probabilities (BPP) using tuf MrBayes (V3.0b4, Huelsenbeck and Ronquist 2001). In the Bayesian inference of the amino acid data, we used the WAG þ G model with Metropolis-coupled Markov chain Mon- te Carlo from a random starting tree. These analyses were run D

psb for 1,000,000 generations with trees sampled each 200 cycles. Four chains were run simultaneously of which three were heated and one was cold, with the initial 20,000 cycles (200 trees) being discarded as the ‘‘burn in.’’ A consensus tree was

C made with the remaining 1800 phylogenies to determine the

psb posterior probabilities at the different nodes. In the MP anal- yses, 1000 bootstrap replicates were analyzed (Felsenstein 1985) with 10 heuristic searches with random-addition-se- quence starting trees and tree bisection-reconnection (TBR)

A branch rearrangements. For the ML bootstrap analysis (500

psb replicates), we used the JTT þ G evolutionary model. For the DNA data set, we used ML and BPP approaches. We used a site-specific GTR model to incorporate different rates in the genes at the three codon positions. This approach B appears to be superior to using a single set of rate parameters psa for protein-coding DNA sequences (Shapiro et al. 2005). Five different rates were used to account for among-site rate var- iation (0.50931, 18S; 0.43367, 16S; 0.60518, 1st codon; 0.16149, 2nd codon; 2.80154, 3rd codon). For the ML analy- A sis, global rearrangements and random sequence addition rep- psa licates (five rounds) were used with TBR branch swapping. The unweighted MP analysis was performed as described above. L rbc RESULTS AND DISCUSSION The seven major lineages of red algae. Seven well- supported lineages were identified within the red al- gae (Figs. 1 and 2). The Cyanidiales was the earliest divergence in the plastid protein tree and was distinct from the remaining red algal lineages (i.e. the Por- phyridiales-(1), Porphyridiales-(2), Porphyridiales- (3), Bangiales, Florideophyceae, and Compsopogon- ales). The Porphyridiales-(2) and the Compsopogon- ales were united in a clade (495% BPP, 55% MP; Fig. GenBankGenBankGenBankGenBankUTEX B 1929 NA NA NA NC_002186 NC_002186 NC_001631 NC_002186 NA NA NC_001631 NC_002186 NC_003386 NC_001631 NC_002186 NC_003386 NC_001631 NC_002186 NC_003386 NC_001631 NC_001675 NC_003386 AY876195 NA NC_001675 NC_003386 AY876199 NC_001675 NA NC_001675 AY876201 NC_001675 NC_001675 AY876226 AY876252 Missing GenBank NA NC_004113 NC_004113 NC_004113 NC_004113 NC_004113 NC_004113 GenBank NA2), NC_000911 whereas NC_000911 NC_000911 NC_000911 NC_000911 the NC_000911 Porphyridiales-(1) grouped together with the monophyletic clade of Bangiales þ Florid- eophyceae (495% BPP, 76% ML; Fig. 1). The exist- (L.) ence of these seven red algal lineages in the plastid sp. PCC BP-1 L. GenBank NAtrees NC_001666 NC_001666 NC_001666is NC_001666 consistent NC_001666 NA with analyses of nuclear SSU rDNA

sp. PCC 7120 GenBank NA(Mu¨ller NC_003272 NC_003272et NC_003272 al. NC_0032722001), NC_003272 NC_003272 plastid SSU rDNA (Oliveira and Bhattacharya 2000), and multi-gene plastid data set polymorpha L. viride Lauterborn Pinus thunbergiana Franco Psilotum nudum Beauvois Zea mays Cyanophora paradoxa Korshikov Glaucocystis nostochinearum Itzigsohn Nostoc Synechocystis 6803 Thermosynechococcus elongatus (Yoon et al. 2002b, 2004). Although we used an extensive concatenated data set of 7PEP (2564 aa) in our analyses, the lineage re-

(Continued). lationships were weakly supported except for the early ller et al. (2003). 1

¨ divergence point of the Cyanidiales and the Bangi- Mu

P1, porphyridiales-(1); P2, porphyridiales-(2); P3,The porphyridiales-(3). accession numbersa of sequences determined in this study are shown in bold text. ales þ Florideophyceae sister group relationship. We ABLE T Taxa Species Source Glaucophyta Cyanobacteria then added 18S and 16S rDNA sequences to the seven- THE MAJOR LINEAGES OF RED ALGAE 487

FIG. 1. Bangiophycean red algal phylo- geny inferred using the maximum likeli- hood (ML) method and the combined plastid protein sequences of PSI P700 chlorophyll a apoprotein A1, PSI P700 chlorophyll a apoprotein A2, PSII reaction center protein D1, PSII 44 KD apoprotein, PSII D2 reaction center protein, RbcL, and TufA. The results of a ML bootstrap analysis are shown above the branches, whereas the values below the branches re- sult from a maximum parsimony bootstrap analysis. The thick branches represent 495% Bayesian posterior probability.

gene DNA sequence data set (9GDS; total 10,463 bp) in on these and our phylogeny, we postulate that an attempt to increase the phylogenetic resolution. after the divergence of the Cyanidiales, the five major Three additional Cyanidiales species were added to red algal lineages radiated around 1200 Ma, likely over test the interrelationship of these taxa and this clade a relatively short evolutionary time period. Thereafter, was used to root the tree. The ML phylogeny tree in- ferred from these data using the site-specific GTR model was similar to the 7PEP tree, except for the po- sition of the Porphyridiales-(3) lineage (Fig. 2). Similar to the 7PEP phylogeny, the Compsopogonales and Porphyridiales-(2) grouped together (55% ML) and the Porphyridiales-(1) showed a weak sister group re- lationship (495% BPP, 53% ML) to the Bangiales and Florideophyceae. Our analyses highlight the inherent difficulties in resolving the intra-lineage relationships of the red algae (except for the position of the Cya- nidiales and the monophyly of the Bangiales þ Florid- eophyceae). This may reflect a rapid radiation of the red algal lineages or poor resolving power of plastid sequences. There are two important fossils to interpret the evolutionary history of the red algae. One is the well- preserved Bangia-like multicellular filamentous , pubescens (Butterfield 2000). This fossil was found from the approximately 1200 million year old (Ma) Hunting Formation in Somerset Island, Can- FIG. 2. Bangiophycean red algal phylogeny inferred using the maximum likelihood (ML) method and the combined DNA ada, and contains putative that indicate sexual sequences of 18S rDNA, 16S rDNA, PSI P700 chlorophyll a differentiation. The second fossil is of Corallinales apoprotein A1, PSI P700 chlorophyll a apoprotein A2, PSII (florideophytes) that contain typical reproductive reaction center protein D1, PSII 44 KD apoprotein, PSII D2 structures from the 599 Ma , reaction center protein, rbcL, and tufA. The results of a ML bootstrap analysis are shown above the branches, whereas the China (Xiao et al. 1998, 2004). Xiao et al. (2004) sug- values below the branches result from a maximum parsimony gested the florideophyte—bangiophyte divergence to bootstrap analysis. The thick branches represent 495% Bayesian have occurred in the Neoproterozoic or earlier. Based posterior probability. 488 HWAN SU YOON ET AL. the Florideophyceae diverged from the ancestor of the Bangiales before 599 Ma when the di- verged from the stem of florideophytes. Yoon et al. (2004) estimated divergence times using molecular clock methods and proposed the following dates for the split of the red algae (1449–1513 Ma), Cyanidiales (1350–1416 Ma), and florideophytes (around 800 Ma, see Fig. 3 in Yoon et al. 2004). Novel findings of our study. Porphyridiales-(2): Por- phyridiales-(2) is comprised of mostly pseudo- filamentous, filamentous, or colonial multicellular taxa (Bangiopsis, Chroodactylon, Chroothece, Kyliniella, Purpureofilum,andStylonema), but also includes uni- cellular forms such as Rhodosorus, Rhodospora, and Rufusia (Fig. 3). Most members of this lineage con- tain sorbitol/digeneaside or only sorbitol as a low mo- lecular weight carbohydrate (Karsten et al. 2003, West et al. 2005). In addition all species of Porphy- ridiales-(2) contain putative group II introns in the psaA gene at conserved positions (H. S. Yoon et al. unpublished data). These ribozymes are unique and are potentially a diagnostic character for this lineage. Group II introns are common in the green algae and in green algal-derived plastids [e.g. and Chlamydomonas (Odom et al. 2004, Perron et al. 2004,

Sheveleva and Hallick 2004)] but apparently are rare FIG. 3. Bangiophycean red algal phylogeny inferred using in the red algae. Our preliminary phylogenetic analy- the maximum likelihood (ML) method and the combined plastid ses of the Pophyridiales-(2) group II introns show that protein sequences of PSI P700 chlorophyll a apoprotein A1 and sequences at the homologous genic site 229 in psaA RbcL. The results of a ML bootstrap analysis are shown above the branches, whereas the values below the branches result from differ significantly in sequence from other introns a maximum parsimony bootstrap analysis. The thick branches present in Flintiella [position 219, Porphyridiales-(3)] represent 495% Bayesian posterior probability. and Rhodella [position 91, Porphyridiales-(1)]. It is like- ly that the introns at position 229 share a single origin (H. S. Yoon et al. unpublished data). This finding sug- gests that these mobile elements invaded the psaAgene Porphyridium are phylogenetically distinct from the (and potentially other sequences) in the plastid gen- Flintiella and Erythrolobus clade. It was previously re- ome of the common ancestor of Porphyridiales-(2) and ported that Flintiella and Porphyridium share the asso- have been maintained in some taxa. ciation of the golgi with the mitochondria and ER Rufusia pilicola diverges first in the Porphyridiales- (Scott et al. 1992) and floridoside as a low molecular (2). This poorly studied species was isolated from weight carbohydrate (Karsten et al. 2003). sloth’s hair by one of the authors (F. D. Ott). The ma- Compsopogonales: This lineage consists of one terial came from the coastal city of Limo´n, Costa Rica freshwater order Compsopogonales and two marine (provided by Dr. Arroyo). The other species of Por- orders Erythropeltidales and Rhodochaetales. Sah- phyridiales-(2) occur in a of habitats, including lingia, Chlidophyllon,andPyrophyllon are included in marine, freshwater, and in caves. Bangiopsis and Kylini- this lineages (Kornmann 1989, Nelson et al. 2003). ella make a well-supported monophyletic group with All of these species are multicellular, though with Purpureofilum that is sister to two Stylonema strains. The varied morphologies. Although our two-gene phylo- SAG stain of S. alsidii formed a well supported a clade geny (Fig. 3) did not support the monophyly of with the UTEX strain. The S. alsidii UTEX strain had Compsopogon spp. with Boldia erythrosiphon (as would been recognized as Goniotrichum elegans (Chauvin) V. be expected; Rintoul et al. 1999, Mu¨ller et al. 2001), May 1965, whereas the SAG strain was renamed from the nuclear SSU rDNA and the plastid multi-gene Goniotrichum alsidii. Because these two S. alsidii strains analyses show a sister group relationship of the Er- show a high sequence divergence (79 substitutions ythropeltidales and Rhodochaetales (Zuccarello et al. among 2610 bp) in the psaAandrbcL genes, a more 2000, Mu¨ller et al. 2001, Yoon et al. 2002b) with the detailed monographic study of Stylonema, including ex- Compsopogonales and B. erythrosiphon. We were, amination of type specimens is required to verify the however unable to isolate the psaA coding region species delimitation. from B. erythrosiphon and only used the rbcL sequence Porphyridiales-(3): The Porphyridiales-(3) lineage in this analysis. Rintoul et al. (1999) suggested that includes the three unicellular genera Erythrolobus, Composopogon coeruleus and Compsopogoniopsis le- Flintiella,andPorphyridium. The three species of ptocladus should be recognized as the single species, THE MAJOR LINEAGES OF RED ALGAE 489

FIG. 4. Red algal phylogeny and alterna- tive taxonomic schemes.

C. coeruleus,basedontheidentityinthesequenceof the Florideophyceae. We did not find any phylo- SSU rDNA and rbcL. genetic evidence to unite the Porphyridiales into a Taxonomic conclusions. A classification system for a single clade (as suggested by Saunders and Hommer- group of organisms should reflect its phylogeny. A sand 2004). This (negative) evidence does not dis- reliable phylogeny ideally uses both broad taxon prove Porphyridiales monophyly. However, it sampling and sufficient phylogenetic information. suggests that if all three or a subset of the lineages This study used analyses of seven- and nine-gene share a monophyletic origin, the branch that unites data sets with selected taxa, and a two-gene data set them may prove difficult to establish unless a great that encompassed most of the bangiophycean genera. deal more (or with higher phylogenetic resolution) Although many clade interrelationships are still un- sequence data is brought to bear on this issue. Our resolved, we clearly identifiedsevenredalgallineag- trees do, however, demonstrate an ancient split of es that should be recognized at the class-level. The taxa formerly classified in the Porphyridiales. For Cyanidiophyceae diverged first in red algal these reasons, we chose to recognize this group as and is separated from the remainder of the red algal three independent clades within the Rhodophytina. lineages. Hence, we propose the creation of two new subphyla, Cyanidiophytina, for this grouping, and Phylum Rhodophyta Wettstein 1901 Rhodophytina that encompasses the rest of the class- 1: Cyanidiophytina H. S. Yoon, K. M. es. Saunders and Hommersand (2004) treated the Mu¨ller, R. G. Sheath, F. D. Ott et D. Bhattacharya, Cyanidiophyceae as the phylum Cyanidiophyta with subphylum nov. another phylum Rhodophyta under the subkingdom Rhodoplantae. However, we believe that it is not ne- Rhodophyta unicellularis, forma globosa vel el- cessary to divide the red algae into two phyla because liptica; habitatio thermae acidae; cum paries this group, including the cyanidiophytes, is phylo- crassus vel paries absens; heterotrophicus vel au- genetically distinct in eukaryotic trees (Rodrı´guez- totophicus; cellulae divisionem vel endospora. Ezpeleta et al. 2005) and they share important bio- Unicellular red algae, spherical or elliptical in chemical features with other red algae such as the shape, inhabiting in acidic and high temperature biosynthesis of floridean starch in the cytoplasm environment; thick wall or lack of ; (Barbier et al. 2005). In addition, all rhodophytes facultative or obligate photo- share an important synapomorphy, a plastid bound autotrophs; cell or endospore form- by two membranes that lacks chl b or c.Rather,our ation. data indicate that it is appropriate to divide the rhodophytes into two subphyla, maintaining the Class Cyanidiophyceae Merola, Castaldo, De traditional characteristics of the phylum as a whole Luca, Gambardella, Musacchio et Taddei 1981 (Fig. 4). Under this taxonomic scheme the subphy- lum Rhodophytina contains six classes: Bangio- Order Cyanidiales Christensen 1962 phyceae, Compsopogonophyceae, Florideophyceae, Family 1 Geitler 1935 , Rhodellophyceae, and Stylo- Genera Cyanidium, Cyanidioschyzon nematophyceae. The interrelationships among the Family 2 Merola, Castaldo, De classes of the subphylum Rhodophytina are unclear Luca, Gambardella, Musacchio et Taddei 1981 except for the monophyly of the Bangiophyceae and Galdieria 490 HWAN SU YOON ET AL.

Subphylum 2: Rhodophytina Unicellular red algae; a single highly lobed plastid with eccentric or centric , Golgi associa- Unicellular, pseudofilamentous or multicellular tion with nucleus and ER; contains mannitol; re- red algae; various plastid morphologies and or- production by cell division. ganellar associations; life histories unknown or Family Rhodellaceae fam. nov. H. S. Yoon, K. M. where known biphasic or triphasic. Mu¨ller, R. G. Sheath, F. D. Ott et D. Bhattacharya, Class 1: Bangiophyceae Wettstein 1901 fam. nov. Characters as for order Order Bangiales Na¨geli 1847 Genera Rhodella, Dixoniella, Glaucosphaera Family Engler 1892 Genera Bangia, Dione, Minerva, Porphyra, Pseudo- Class 6: Stylonematophyceae H.S. Yoon, K.M. bangia Mu¨ller, R.G. Sheath, F.D. Ott et D. Bhattacharya, classis nov. Class 2: Compsopogonophyceae G. W. Saunders et Hommersand 2004 Rhodophyta unicelluaris, pseudofilamentosis vel filamentosis; chloroplasti cum morphologiae Order 1 Compsopogonales Skuja 1939 variabilis et cum vel sine pyrenoides; Golgi cum Family 1 Boldiaceae Herndon 1964 reticulo endoplasmatico et conso- Genus Boldia ciatus; reproductio a division cellulosa vel mono- Family 2 Compsopogonaceae Schmitz in Engler spora. et Prantl 1896 Unicellular or pseudofilamentous or filamentous Genus Compsopogon red algae; various plastid morphologies with or Order 2 Erythropeltidales Garbary, Hansen et without pyrenoid; Golgi association with mito- Scagel 1980 and ER; reproduction by cell division Family G. M. Smith 1933 or monospores. Genera Erythrotrichia, Chlidophyllon, Erythrocladia, Order Stylonematales Drew 1956 Pyrophyllon, Sahlingia Family Drew 1956 Order 3 Rhodochaetales Bessey 1907 Genera Stylonema, Bangiopsis, Chroodactylon, Family Rhodochaetaceae Schmitz in Engler et Chroothece, Purpureofilum, Rhodosorus, Rhodospora, Prantl 1896 Rufusia Genus Rhodochaete This work was supported by grants from the National Science Class 3: Florideophyceae Cronquist 1960 Foundation awarded to D. B., K. M. M., and R. G. S. (DEB Multiple orders. 01-07754) and to D. B. (MCB 02-36631). Class 4: Porphyridiophyceae H. S. Yoon, K. M. Mu¨ller, R. G. Sheath, F. D. Ott et D. Bhattacharya, Adl, S. M., Simpson, A. G., Farmer, M. A., Andersen, R. A., class nov. Anderson, O. R., Barta, J. R., Bowser, S. S., Brugerolle, G., Fensome, R. A., Fredericq, S., James, T. Y., Karpov, S., Rhodophyta unicellularis; chloroplasti singularis Kugrens, P., Krug, J., Lane, C. E., Lewis, L. A., Lodge, J., et ramosi vel stellaris cum pyrenoides; Golgi cum Lynn, D. H., Mann, D. G., McCourt, R. M., Mendoza, L., reticulo endoplasmatico et mitochondrion conso- Moestrup, O., Mozley-Standridge, S. E., Nerad, T. A., Shearer, ciatus; cellulae cum floridoside; reproductio a C. A., Smirnov, A. V., Spiegel, F. W. & Taylor, M. F. 2005. The new higher level classification of with emphasis on divisio cellulosa. the of . J. Eukaryot. Microbiol. 52:399–451. Unicellular red algae with a single branched or Barbier, G., Oesterholt, C., Larson, M. D., Halgren, R. G., Wilker- stellate plastid with or without pyrenoid; Golgi son, C., Garavito, R. M., Benning, C. & Weber, A. P. M. 2005. association with mitochondria and ER; cells with Comparative genomics of two closely related unicellular floridoside as a low molecular weight carbohy- thermo-acidophilic red algae, Galdieria sulphuraria and Cyanid- ioschyzon merolae, reveals the molecular basis of the metabolic drate; reproduction by cell division. flexibility of Galdieria sulphuraria and significant differences in Order Porphyridiales Kylin ex Skuja 1939 carbohydrate of both algae. Plant Physiol. 137: Family Porphyridiaceae Skuja 1939 440–74. Genera Porphyridium, Erythrolobus, Flintiella Bhattacharya, D. & Medlin, L. 1995. The phylogeny of plastids: a review based on comparisons of small-subunit ribosomal RNA Class 5: Rhodellophyceae Cavalier-Smith 1998 coding regions. J. Phycol. 31:489–98. Bhattacharya, D., Yoon, H. S. & Hackett, J. D. 2004. Photosynthetic Order Rhodellales ord. nov. H. S. Yoon, K. M. eukaryotes unite: endosymbiosis connects the dots. BioEssays 26:50–60. Mu¨ller, R. G. Sheath, F. D. Ott et D. Bhattacharya, Burger, G., Saint-Louis, D., Gray, M. W. & Lang, B. F. 1999. Com- order nov. plete sequence of the mitochondrial DNA of the red alga Rhodophyta unicelluaris; chloroplasti singularis Porphyra purpurea. Cyanobacterial introns and shared ancestry et lobati cum pyrenoides in centro vel a centro; of red and green algae. Plant Cell 11:1675–94. Butterfield, N. J. 2000. Bangiomorpha pubescens n. gen., n. sp.: im- Golgi cum reticulo endoplasmatico et nucleus plications for the evolution of , multicellularity, and the consociatus; cum mannitol; reproductio a divisio Mesoproterozoic/Neoproterozoic radiation of eukaryotes. cellulosa. Paleobiology 26:386–404. THE MAJOR LINEAGES OF RED ALGAE 491

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